The long-term goal of this project is to dissect the pathogenesis of Cryptococcus meningitis. In recent years infection by Cryptococcus neoformans has increased considerably, particularly in immunocomprised individuals and AIDS patients. This pathogen has a predilection to the brain, resulting in devastating meningoencephalitis. It is unclear how C. neoformans traverses across the blood-brain barrier and causes the central nervous system infection. We propose to investigate the mechanisms of C. neoformans invasion in human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. We have established in vitro and in vivo BBB models for the pathogen invasion studies. Using these model systems, we found that C. neoformans, either treated with hyaluronidase or with 4-methylumbelliferone (a hyaluronan synthase inhibitor) substantially reduced its binding ability to HBMEC. In contrast, exogenous hyaluronan was found to stimulate C. neoformans binding activity. The CPS1 protein sequence shares homology with hyaluronan synthase. The cpsl-disrupted strain lost its hyaluronan on the cell surface, and also substantially reduced its binding activity to HBMEC. Finally, anti-CD44 blocking antibody could neutralize the interaction between C. neoformans and HBMEC. Taken together, our preliminary studies suggested that the C. neoformans capsule component, hyaluronan, and HBMEC CD44 were involved in the invasion process. In this proposal, we will test the hypothesis that C. neoformans hyaluronan and HBMEC CD44 are crucial for C. neoformans pathogenesis. We will explore the mechanisms of C. neoformans invasion to HBMEC by the following Aims: First, we will examine the role of CPS1 in hyaluronan synthesis and C. neoformans invasion. Second, we will determine how C. neoformans hyaluronan contribute to the binding and invasion of HBMEC. Third, we will determine whether and how HBMEC CD44 functions as a HA receptor during C. neoformans invasion. The proposed approach will allow us to determine the role(s) of the crucial C. neoformans component, hyaluronan, and host responses during invasion. The information derived from this proposal should be helpful in the development of novel strategies to prevent C. neoformans meningitis and its associated morbidity.